Methods for producing enriched populations of human retinal pigment epithelium cells

ABSTRACT

This invention relates to methods for improved cell-based therapies for retinal degeneration and for differentiating human embryonic stem cells and human embryo-derived into retinal pigment epithelium (RPE) cells and other retinal progenitor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.11/041,382, filed Jan. 24, 2005, which claims the benefit of U.S.Provisional Application No. 60/538,964, filed Jan. 23, 2004, thecontents of which are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

This invention relates generally to methods for improved cell-basedtherapies for retinal degeneration and other visual disorders as well astreatment of Parkinson's disease and for differentiating mammalianembryonic stem cells and mammalian embryo-derived cells into retinalpigment epithelium (RPE) cells and other eye tissue including, but notlimited to, rods, cones, bipolar, corneal, neural, iris epithelium, andprogenitor cells.

BACKGROUND OF THE INVENTION

Many parts of the central nervous system (CNS) exhibit laminarorganization, and neuropathological processes generally involve morethan one of these multiple cellular layers. Diseases of the CNSfrequently include neuronal cell loss, and, because of the absence ofendogenous repopulation, effective recovery of function followingCNS-related disease is either extremely limited or absent. Inparticular, the common retinal condition known as age-related maculardegeneration (AMD) results from the loss of photoreceptors together withthe retinal pigment epithelium (RPE), with additional variableinvolvement of intenuncial (“relay”) neurons of the inner nuclear layer(INL). Restoration of moderate-to-high acuity vision, therefore,requires the functional replacement of some or all of the damagedcellular layers.

Anatomically, retinitis pigmentosa (RP), a family of inherited retinaldegenerations, is a continuing decrease in the number of photoreceptorcells which leads to loss of vision. Although the phenotype is similaracross most forms of RP, the underlying cellular mechanisms are diverseand can result from various mutations in many genes. Most involvemutations that alter the expression of photoreceptor-cell-specificgenes, with mutations in the rhodopsin gene accounting for approximately10% of these. In other forms of the disease, the regulatory genes ofapoptosis are altered (for example, Bax and Pax2). AMD is a clinicaldiagnosis encompassing a range of degenerative conditions that likelydiffer in etiology at the molecular level. All cases of AMD share thefeature of photoreceptor cell loss within the central retina. However,this common endpoint appears to be a secondary consequence of earlierabnormalities at the level of the RPE, neovascularization, andunderlying Bruch's membrane. The latter may relate to difficulties withphotoreceptor membrane turnover, which are as yet poorly understood.Additionally, the retinal pigment epithelium is one of the mostimportant cell types in the eye, as it is crucial to the support of thephotoreceptor function. It performs several complex tasks, includingphagocytosis of shed outer segments of rods and cones, vitamin Ametabolism, synthesis of mucoploysacharides involved in the metaboliteexchange in the subretinal space, transport of metabolites, regulationof angiogenesis, absorption of light, enhancement of resolution ofimages, and the regulation of many other functions in the retina throughsecreted proteins such as proteases and protease inhibitors.

An additional feature present in some cases of AMD is the presence ofaberrant blood vessels, which result in a condition known as choroidalneovascularization (CNV). This neovascular (“wet”) form of AMD isparticularly destructive and seems to result from a loss of properregulation of angiogenesis. Breaks in Bruch's membrane as a result ofRPE dysfunction allows new vessels from the choroidal circulation accessto the subretinal space, where they can physically disrupt outer-segmentorganization and cause vascular leakage or hemorrhage leading toadditional photoreceptor loss.

CNV can be targeted by laser treatment. Thus, laser treatment for the“wet” form of AMD is in general use in the United States. There areoften undesirable side effects, however, and therefore patientdissatisfaction with treatment outcome. This is due to the fact thatlaser burns, if they occur, are associated with photoreceptor death andwith absolute, irreparable blindness within the corresponding part ofthe visual field. In addition, laser treatment does not fix theunderlying predisposition towards developing CNV. Indeed, laser burnshave been used as a convenient method for induction of CNV in monkeys(Archer and Gardiner, 1981). Macular laser treatments for CNV are usedmuch more sparingly in other countries such as the U.K. There is nogenerally recognized treatment for the more common “dry” form of AMD, inwhich there is photoreceptor loss overlying irregular patches of RPEatrophy in the macula and associated extracellular material calleddrusen.

Since RPE plays an important role in photoreceptor maintenance, andregulation of angiogenesis, various RPE malfunctions in vivo areassociated with vision-altering ailments, such as retinitis pigmentosa,RPE detachment, displasia, atrophy, retinopathy, macular dystrophy ordegeneration, including age-related macular degeneration, which canresult in photoreceptor damage and blindness. Specifically and inaddition to AMD, the variety of other degenerative conditions affectingthe macula include, but are not limited to, cone dystrophy, cone-roddystrophy, malattia leventinese, Doyne honeycomb dystrophy, Sorsby'sdystrophy, Stargardt disease, pattern/butterfly dystrophies, Bestvitelliform dystrophy, North Carolina dystrophy, central areolarchoroidal dystrophy, angioid streaks, and toxic maculopathies.

General retinal diseases that can secondarily affect the macula includeretinal detachment, pathologic myopia, retinitis pigmentosa, diabeticretinopathy, CMV retinitis, occlusive retinal vascular disease,retinopathy of prematurity (ROP), choroidal rupture, ocularhistoplasmosis syndrome (POHS), toxoplasmosis, and Leber's congenitalamaurosis. None of the above lists is exhaustive.

All of the above conditions involve loss of photoreceptors and,therefore, treatment options are few and insufficient.

Because of its wound healing abilities, RPE has been extensively studiedin application to transplantation therapy. In 2002, one year into thetrial, patients were showing a 30-50% improvement. It has been shown inseveral animal models and in humans (Gouras et al., 2002, Stanga et al.,2002, Binder et al., 2002, Schraermeyer et al., 2001, reviewed by Lundet al., 2001) that RPE transplantation has a good potential of visionrestoration. However, even in an immune-privileged site such as the eye,there is a problem with graft rejection, hindering the progress of thisapproach if allogenic transplantation is used. Although newphotoreceptors (PRCs) have been introduced experimentally bytransplantation, grafted PRCs show a marked reluctance to link up withsurviving neurons of the host retina. Reliance on RPE cells derived fromfetal tissue is another problem, as these cells have shown a very lowproliferative potential. Emory University researchers performed a trialwhere they cultured RPE cells from a human eye donor in vitro andtransplanted them into six patients with advanced Parkinson's Disease.Although a 30-50% decrease in symptoms was found one year aftertransplantation, there is a shortage of eye donors, this is not yet FDAapproved, and there would still exist a need beyond what could be met bydonated eye tissue.

Thus far, therapies using ectopic RPE cells have been shown to behavelike fibroblasts and have been associated with a number of destructiveretinal complications including axonal loss (Villegas-Perez, et al,1998) and proliferative vitreoretinopathy (PVR) with retinal detachment(Cleary and Ryan, 1979). RPE delivered as a loose sheet tends to scrollup. This results in poor effective coverage of photoreceptors as well asa multilayered RPE with incorrect polarity, possibly resulting in cystformation or macular edema.

Delivery of neural retinal grafts to the subretinal (submacular) spaceof the diseased human eye has been described in Kaplan et al. (1997),Humayun et al. (2000), and del Cerro et al. (2000). A serious problemexists in that the neural retinal grafts typically do not functionallyintegrate with the host retina. In addition, the absence of an intactRPE monolayer means that RPE dysfunction or disruption of Bruch'smembrane has not been rectified. Both are fundamental antecedents ofvisual loss.

Thus, there exists no effective means for reconstituting RPE in any ofthe current therapies and there remain deficiencies in each,particularly the essential problem of a functional disconnection betweenthe graft and the host retina. Therefore there exists the need for animproved retinal therapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide improved methods forthe derivation of eye cells including, but not limited to, neural cells,including horizontal cells and amacrine cells, retinal cells such asrods and cones, corneal cells, vascular cells, and RPE and RPE-likecells from stem cells and to provide improved methods and therapies forthe treatment of retinal degeneration. In particular, these methodsinvolve the use of RPE and RPE-like cells derived from human embryonicstem cells.

One embodiment of the present invention provides an improved method ofgenerating cells for therapy for retinal degeneration using RPE cells,RPE-like cells, the progenitors of these cells or a combination of twoor three of any of the preceding derived from mammalian embryonic stemcells in order to treat various conditions including but not limited toretinitis pigmentosa and macular degeneration and associated conditions.The cell types which can be produced using this invention include, butare not limited to, RPE, RPE-like cells, and RPE progenitors. Cellswhich may also be produced include iris pigmented epithelial (IPE)cells. Vision associated neural cells including intemuncial neurons(e.g. “relay” neurons of the inner nuclear layer (INL)) and amacrinecells (interneurons that interact at the second synaptic level of thevertically direct pathways consisting of thephotoreceptor-bipolar-ganglion cell chain—they are synaptically activein the inner plexiform layer (IPL) and serve to integrate, modulate andinterpose a temporal domain to the visual message presented to theganglion cell) can also be produced using this invention. Additionally,retinal cells, rods, cones, and corneal cells can be produced. In afurther embodiment of the present invention, cells providing thevasculature of the eye can also be produced. The cells of the presentinvention may be transplanted into the subretinal space by usingvitrectomy surgery. Non-limiting examples include the transplantation ofthese cells in a suspension, matrix, or substrate. Animal models ofretinitis pigmentosa that may be treated include rodents (rd mouse,RPE-65 knockout mouse, tubby-like mouse, RCS rat, cats (Abyssinian cat),and dogs (cone degeneration “cd” dog, progressive rod-cone degeneration“prcd” dog, early retinal degeneration “erd” dog, rod-cone dysplasia 1,2 & 3 “rcd1, rcd2 & rcd3” dogs, photoreceptor dysplasia “pd” dog, andBriard “RPE-65” (dog). Evaluation is performed using behavioral tests,fluorescent angiography, histology, or functional testing such asmeasuring the ability of the cells to perform phagocytosis(photoreceptor fragments), vitamin A metabolism, tight junctionsconductivity, or evaluation using electron microscopy. One of the manyadvantages to the methods presented here is the ability to produce andtreat many more patients than it would be possible to treat if one werelimited to using eye donor tissue.

A further embodiment of the present invention provides methods for thespontaneous differentiation of hES cells into cells with numerouscharacteristics of RPE. These RPE preparations are capable of phenotypicchanges in culture and maintaining RPE characteristics through multiplepassages. The present invention also provides for methods ofdifferentiation of established RPE cell lines into alternate neuronallineages, corneal cells, retinal cells as a non-limiting example throughthe use of bFGF or FGF.

Another embodiment of the present invention is a method for thederivation of new RPE lines and progenitor cells from existing and newES cell lines. There can be variations in the properties, such as growthrate, expression of pigment, or de-differentiation andre-differentiation in culture, of RPE-like cells when they are derivedfrom different ES cell lines. There can be certain variations in theirfunctionality and karyotypic stability, so it is desirable to providemethods for the derivation of new RPE lines and new ES cell lines whichwould allow choosing the lines with desired properties that can beclonally selected to produce a pure population of high quality RPE-likecells.

In yet another embodiment, the present invention provides an isolatedRPE or RPE-like cell line which varies from established RPE cell linesin at least one of the characteristics selected from the groupconsisting of: growth rate, expression of pigment, de-differentiation inculture, and re-differentiation in culture.

Cells which may also be derived from existing and new ES cell linesinclude iris pigmented epithelial (IPE) cells. In an additionalembodiment, vision associated neural cells including internuncialneurons (e.g. “relay” neurons of the inner nuclear layer (INL)) andamacrine cells can also be produced using this invention. Additionally,retinal cells, rods, cones, and corneal cells can be produced. In afurther embodiment of the present invention, cells providing thevasculature of the eye can also be produced.

Another embodiment of the present invention is a method for thederivation of RPE lines or precursors to RPE cells that have anincreased ability to prevent neovascularization. Such cells can beproduced by aging a somatic cell from a patient such that telomerase isshortened where at least 10% of the normal replicative lifespan of thecell has been passed, then the use of said somatic cell as a nucleartransfer donor cell to create cells that overexpress angiogenesisinhibitors such as Pigment Epithelium Derived Factor (PEDF/EPC-1).Alternatively such cells may be genetically modified with exogenousgenes that inhibit neovascularization.

Another embodiment of the present invention utilized a bank of ES orembryo-derived cells with homozygosity in the HLA region such that saidcells have reduced complexity of their HLA antigens.

Therefore, an additional embodiment of the present invention includesthe characterization of ES-derived RPE-like cells. Although theES-derived pigmented epithelial cells strongly resemble RPE by theirmorphology, behavior and molecular markers, their therapeutic value willdepend on their ability to perform RPE functions and to remainnon-carcinogenic. Therefore, the ES-derived RPE cells are characterizedusing one or more of the following techniques: (i) assessment of theirfunctionality, i.e. phagocytosis of the photoreceptor fragments, vitaminA metabolism, wound healing potential; (ii) evaluation of thepluripotency of RPE-like ES cells derivatives through animal modeltransplantations, (as a non-limiting example this can include SCIDmice); (iii) phenoytping and karyotyping of RPE-like cells; (iv)evaluation of ES cells-derived RPE-like cells and RPE tissue by geneexpression profiling, (v) evaluation of the expression of molecularmarkers of RPE at the protein level, including bestrophin, CRALBP,RPE-65, PEDF, and the absence of ES markers, and (vi) evaluation of theratio of RPE and neural markers. The cells can also be evaluated basedon their expression of transcriptional activators normally required forthe eye development, including rx/rax, chx10/vsx-2/alx, ots-1, otx-2,six3/optx, six6/optx2, mitf, pax6/mitf, and pax6/pax2 (Fischer and Reh,2001, Baumer et al., 2003).

An additional embodiment of the present invention is a method for thecharacterization of ES-derived RPE-like cells using at least one of thetechniques selected from the group consisting of (i) assessment of theES-derived RPE-like cells functionality; (ii) evaluation of thepluripotency of RPE-like ES cell derivatives through animal modeltransplantations; (iii) phenoytping and karyotyping of RPE-like cells;(iv) evaluation of gene expression profiling, (v) evaluation of theexpression of molecular markers of RPE at the protein level; and (vi)the expression of transcriptional activators normally required for theeye development. In a further embodiment these techniques may be usedfor the assessment of multiple hES cell-derived cell types.

Another embodiment of the present invention is a method for thederivation of RPE cells and RPE precursor cells directly from human andnon-human animal morula or blastocyst-staged embryos (EDCs) without thegeneration of ES cell lines.

Embryonic stem cells (ES) can be indefinitely maintained in vitro in anundifferentiated state and yet are capable of differentiating intovirtually any cell type. Thus human embryonic stem (hES) cells areuseful for studies on the differentiation of human cells and can beconsidered as a potential source for transplantation therapies. To date,the differentiation of human and mouse ES cells into numerous cell typeshave been reported (reviewed by Smith, 2001) including cardiomyocytes[Kehat et al. 2001, Mummery et al., 2003 Carpenter et al., 2002],neurons and neural precursors (Reubinoff et al. 2000, Carpenter et al.2001, Schuldiner et al., 2001), adipocytes (Bost et al., 2002, Aubert etal., 1999), hepatocyte-like cells (Rambhatla et al., 2003), hematopoeticcells (Chadwick et al., 2003). oocytes (Hubner et al., 2003),thymocyte-like cells (Lin R Y et al., 2003), pancreatic islet cells(Kahan, 2003), and osteoblasts (Zur Nieden et al., 2003). Anotherembodiment of the present invention is a method of identifying cellssuch as RPE cells, hematopoietic cells, muscle cells, liver cells,pancreatic beta cells, neurons, endothelium, progenitor cells or othercells useful in cell therapy or research, derived from embryos,embryonic stem cell lines, or other embryonic cells with the capacity todifferentiate into useful cell types by comparing the messenger RNAtranscripts of such cells with cells derived in-vivo. This methodfacilitates the identification of cells with a normal phenotype and forderiving cells optimized for cell therapy for research.

The present invention provides for the differentiation of human ES cellsinto a specialized cell in the neuronal lineage, the retinal pigmentepithelium (RPE). RPE is a densely pigmented epithelial monolayerbetween the choroid and neural retina. It serves as a part of a barrierbetween the bloodstream and retina, and it's functions includephagocytosis of shed rod and cone outer segments, absorption of straylight, vitamin A metabolism, regeneration of retinoids, and tissuerepair. (Grierson et al., 1994, Fisher and Reh, 2001, Marmorstein etal., 1998). The RPE is easily recognized by its cobblestone cellularmorphology of black pigmented cells. In addition, there are severalknown markers of the RPE, including cellular retinaldehyde-bindingprotein (CRALBP), a cytoplasmic protein that is also found in apicalmicrovilli (Bunt-Milam and Saari, 1983); RPE65, a cytoplasmic proteininvolved in retinoid metabolism (Ma et al., 2001, Redmond et al., 1998);bestrophin, the product of the Best vitelliform macular dystrophy gene(VMD2, Marmorstein et al., 2000), and pigment epithelium derived factor(PEDF) a 48 kD secreted protein with angiostatic properties (Karakousiset al., 2001, Jablonski et al., 2000).

An unusual feature of the RPE is its apparent plasticity. RPE cells arenormally mitotically quiescent, but can begin to divide in response toinjury or photocoagulation. RPE cells adjacent to the injury flatten andproliferate forming a new monolayer (Zhao et al, 1997). Several studieshave indicated that the RPE monolayer can produce cells of fibroblastappearance that can later revert to their original RPE morphology(Grierson et al., 1994, Kirchhof et al., 1988, Lee et al., 2001). It isunclear whether the dividing cells and pigmented epithelial layer arefrom the same lineage as two populations of RPE cells have beenisolated: epithelial and fusiforms. (McKay and Burke, 1994). In vitro,depending on the combination of growth factors and substratum, RPE canbe maintained as an epithelium or rapidly dedifferentiate and becomeproliferative (Zhao 1997, Opas and Dziak, 1994). Interestingly, theepithelial phenotype can be reestablished in long-term quiescentcultures (Griersion et al., 1994).

In mammalian development, RPE shares the same progenitor with neuralretina, the neuroepithelium of the optic vesicle. Under certainconditions, it has been suggested that RPE can transdifferentiate intoneuronal progenitors (Opas and Dziak, 1994), neurons (Chen et al., 2003,Vinores et al., 1995), and lens epithelium (Eguchi, 1986). One of thefactors which can stimulate the change of RPE into neurons is bFGF (Opazand Dziak, 1994, a process associated with the expression oftranscriptional activators normally required for the eye development,including rx/rax, chx10/vsx-2/alx, ots-1, otx-2, six3/optx, six6/optx2,mitf, and pax6/pax2 (Fischer and Reh, 2001, Baumer et al., 2003).Recently, it has been shown that the margins of the chick retina containneural stem cells (Fischer and Reh, 2000) and that the pigmented cellsin that area, which express pax6/mitf, can form neuronal cells inresponse to FGF (Fisher and Reh, 2001).

The present invention provides for the derivation of trabecular meshworkcells from hES and also for genetically modified trabecular meshworkcells for the treatment of glaucoma.

The present invention also provides for the derivation of trabecularmeshwork cells from RPE progenitors and RPE-like cells and also forgenetically modified trabecular meshwork cells for the treatment ofglaucoma.

In another embodiment, the present invention provides a method forisolating RPE-like cells. Such a method may comprise: a) culturing hEScells in medium that supports proliferation and transdifferentiation ofhES cells to RPE-like cells; b) selecting the cells of step a) thatexhibit the signs of differentiation along the neural lineage; c)passaging the cells selected in step b) using an enzyme, such as a or acombination of collagenase(s) and/or a dissociation buffer (non-limitingexamples of these include trypsin, collagenase IV, collagenase I,dispase, EDTA, or other commercially available dissociation buffers)until pigmented epithelial islands appear or multiply in number; and d)selecting pigmented or non-pigmented cells passaged in step c) forestablishment of high purity RPE-like cultures. In certain aspects, thehES cells of the invention may be cultured in any medium that supportsproliferation and transdifferentiation. In other aspects, the hES cellsare cultured in medium that contains Serum Replacement. In a specificaspect, the hES cells of the invention may be cultured in medium thatincludes knockout high glucose DMEM supplemented with 500 u/mlPenicillin, 500 μg/ml streptomycin, 1% non-essential amino acidssolution, 2 mM GlutaMAX I, 0.1 mM beta-mercaptoethanol, 4-80 ng/ml bFGF,and 8.4%-20% Serum Replacement. Optionally, the hES cells of the presentinvention are cultured in medium that further comprises 10-100 ng/mlhuman LIF. Optionally, the hES culture medium of the invention furthercomprises Plasmanate. Plasmanate may be added to a final concentrationof about 1% to about 25% (e.g., about 1%, 4%, 6%, 8%, 12%, 16% or 20%).In another aspect, the hES cells of the invention may be passagedrepeatedly, including 2, 3, 5, 7, 10 or more times. Differentiatingcells may be selected due to their expression of neural-lineage specificmarkers. Exemplary neural-lineage specific markers include and Pax6. Ina preferred embodiment bFGF is added to the RPE cultures duringproliferation and the cells are cultured without bFGF duringdifferentiation.

The present invention includes methods for the derivation of RPE cellsand RPE precursor cells directly from human and non-human animal morulaor blastocyst-staged embryos (EDCs) without the generation of ES celllines. In one embodiment, such a method comprises the steps of: a)maintaining ES cells in vitro in an undifferentiated state; b)differentiating the ES cells into RPE and RPE precursor cells; c)identifying the RPE cells by comparing the messenger RNA transcripts ofsuch cells with cells derived in-vivo and/or identifying the RPE cellsby comparing the protein expression profile with known RPE cells and/orphenotypic assessment; and e) identifying and/or isolating RPE cellsand/or RPE precursors.

Further provided by the present invention are methods for the derivationof RPE lines or precursors to RPE cells that have an increased abilityto prevent neovascularization, said methods comprising: a) aging asomatic cell from an animal such that telomerase is shortened wherein atleast 10% of the normal replicative lifespan of the cell has beenpassed; and, b) using the somatic cell as a nuclear transfer donor cellto create cells that overexpress angiogenesis inhibitors, wherein theangiogenesis inhibitors can be Pigment Epithelium Derived Factor(PEDF/EPC-1).

The present invention provides methods for the treatment of Parkinson'sdisease with hES cell-derived RPE, RPE-like and/or RPE progenitor cells.These may be delivered by stereotaxic intrastriatal implantation with ormicrocarriers. Alternately, they may be delivered without the use ofmicrocarriers. The cells may also be expanded in culture and used in thetreatment of Parkinson's disease by any method known to those skilled inthe art.

Other features and advantages of the invention will be apparent from thefollowing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F. is a series of photographs showing the appearance ofpigmented areas (characteristic of RPE cells) in spontaneouslydifferentiating hES cells. FIG. 1A is a photograph of pigmented regionsin a 2.5 month old adherent culture, a well of a 4-well plate, scanned;FIG. 1B is a photograph of pigmented regions in a 2.5 month old culturesgrown as EB; at 45× magnification; FIG. 1C is a photograph of apigmented area of an adherent culture; FIG. 1D is a photograph of apigmented region of an EB; FIG. 1E is a photograph of the boundarybetween pigmented region and the rest of the culture, ×200; Figure Fsame as Figure E but at ×400 magnification. Arrows in A and B point topigmented regions.

FIG. 2A-F. is a series of photographs which show the loss and regain ofpigmentation and epithelial morphology in culture. FIG. 2A is aphotograph showing primary EB outgrowth, 1 week; FIG. 2B is a photographshowing the primary culture of cells, hand-picked, 1 week; FIG. 2C is aphotograph showing epithelial islet surrounded by proliferating cells;FIG. 2D is a photograph showing the regain of pigmentation andepithelial morphology in 1 month old culture; FIG. 2E is a photographshowing the culture after 3 passages, ×200 magnification; FIG. 2F showsthe same culture as in E, ×400 magnification, Hoffman microscopy. Blackarrows point to pigmented cells, white arrows show outgrowing cells withno pigment.

FIG. 3 Left Panel (A-D) and Right Panel is a series of photographs andone graph—these show markers of RPE in hES cells-derived pigmentedepithelial cells. FIGS. 3A and 3B are photographs showingimmunolocalization of RPE marker, bestrophin and corresponding phasemicroscopy field, ×200 magnification; FIGS. 3C and 3D are photographsshowing CRALBP and corresponding phase contrast microscopy field, ×400magnification. Arrows show the colocalization of bestrophin (A) andCRALBP (C) to pigmented cells (C, D); arrowheads point to the absence ofstaining for these proteins (A, B) in non-pigmented regions (C, D).

FIG. 3, Right Panel (top) shows a photograph of Western blot of celllysates with antibodies to bestrophin (a) and CRALBP (b); (c),(d)—undifferentiated hES cells, c—control to anti-CRALBP antibody,d—control to anti-bestrophin antibody

FIG. 3, Right Panel (bottom) shows a comparison of RPE65 expression inmature and immature RPE-like cells by real-time RT-PCR. Sample numbers1, 6 and 7 are mature seven-weeks old culture; sample numbers 2, 3 4 and5 are immature fifteen-days old cultures; and sample number 8 isundifferentiated hES cells.

FIG. 4 shows photographs which demonstrate the expression of markers ofPax6 (FIG. 4A), Pax2 (FIG. 4E) and mitf (FIG. 4B, FIG. 4F) in RPE-likecells in long-term quiescent cultures. FIG. 4C, FIG. 4G—phase contrast,FIG. 4D, FIG. 4H—merged images of Pax6/mitf/phase contrast (FIG. 4A,FIG. 4B, FIG. 4C) and Pax2/mitf/phase contrast (FIG. 4E, FIG. 4F, FIG.4G).

FIG. 5A-B show photographs of RPE differentiation in the culture ofhuman embryo-derived cells bypassing the stage of derivation of ES celllines.

FIG. 6 shows the transcriptional comparison of RPE preparations. FIG.6A-F—Based on the Ontological annotation, this table represents theexpression patterns of RPE related genes for hES cell-derived retinalpigment epithelium (hES-RPE), hES cell derived transdifferentiated(hES-RPE-TD), ARPE-19 and D407, and freshly isolated human RPE (fe-RPE).FIG. 6G—Further data mining revealed known RPE specific ontologies, suchas melanin biosynthesis, vision, retinol-binding, only in fetal RPE andES-RPE but not ARPE-19.

FIG. 7 shows generation of neural progenitors from hES cells. A)Overgrown culture of hES cell, stereomicroscopy. B) Spheroids (arrow inA) were removed and plated onto gelatin-coated plates in EB medium,producing spindle-like cells in 1-2 weeks. C), D) Staining of the cellsshown in B) with antibodies to tubulin beta III (C) and nestin (D).Magnification: A), ×60; B-D), ×200.

FIG. 8 shows morphology of different RPE cultures. A) Uniformdifferentiated RPE. B) Some elongated non-pigmented cells (arrows). C)Pigmented islands surrounded by non-pigmented cells, the culturedescribed as a candidate for hand-picking of the pigmented cells aftercollagenase and/or dispase digestion. D) Transdifferentiated cells.Magnification ×200.

FIG. 9 is a series of photographs showing the appearance of rod andcone-like structures in differtiating cultures of hES cells. FIG. 9 a isa histological examination of differentiating cultures, stained withhematoxylin-eosin, ×200. FIGS. 9 b-9 e are RT-PCR analyses of Opsin 5and Opsin 1 (9 b), recoverin (9 c), rhodopsin (9 d) and Keratin 12 (9 e)in these cultures.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are described in detail and may befurther illustrated by the provided examples. As used in the descriptionherein and throughout the claims that follow, the meaning of “a,” “an,”and “the” includes plural reference unless the context clearly dictatesotherwise. Also, as used in the description herein, the meaning of “in”includes “in” and “on” unless the context clearly dictates otherwise.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitioner indescribing the compositions and methods of the invention and how to makeand use them. For convenience, certain terms may be highlighted, forexample using italics and/or quotation marks. The use of highlightinghas no influence on the scope and meaning of a term; the scope andmeaning of a term is the same, in the same context, whether or not it ishighlighted. It will be appreciated that the same thing can be said inmore than one way. Consequently, alternative language and synonyms maybe used for any one or more of the terms discussed herein, nor is anyspecial significance to be placed upon whether or not a term iselaborated or discussed herein. Synonyms for certain terms are provided.A recital of one or more synonyms does not exclude the use of othersynonyms. The use of examples anywhere in this specification, includingexamples of any terms discussed herein, is illustrative only, and in noway limits the scope and scope of the invention so long as data areprocessed, sampled, converted, or the like according to the inventionwithout regard for any particular theory or scheme of action.

DEFINITIONS

By “embryo” or “embryonic” is meant a developing cell mass that has notimplanted into the uterine membrane of a maternal host. An “embryoniccell” is a cell isolated from or contained in an embryo. This alsoincludes blastomeres, obtained as early as the two-cell stage, andaggregated blastomeres.

The term “embryonic stem cells” refers to embryo-derived cells. Morespecifically it refers to cells isolated from the inner cell mass ofblastocysts or morulae and that have been serially passaged as celllines.

The term “human embryonic stem cells” (hES cells) refers humanembryo-derived cells. More specifically hES refers to cells isolatedfrom the inner cell mass of human blastocysts or morulae and that havebeen serially passaged as cell lines and can also include blastomeresand aggregated blastomeres.

The term “human embryo-derived cells” (hEDC) refers to morula-derivedcells, blastocyst-derived cells including those of the inner cell mass,embryonic shield, or epiblast, or other totipotent or pluripotent stemcells of the early embryo, including primitive endoderm, ectoderm, andmesoderm and their derivatives, also including blastomeres and cellmasses from aggregated single blastomeres or embryos from varying stagesof development, but excluding human embryonic stem cells that have beenpassaged as cell lines.

Embryonic stem (ES) cells which have the ability to differentiate intovirtually any tissue of a human body can provide a limitless supply ofrejuvenated and histocompatible cells for transplantation therapy, asthe problem of immune rejection can be overcome with nuclear transferand parthenogenetic technology. The recent findings of Hirano et al(2003) have shown that mouse ES cells can produce eye-like structures indifferentiation experiments in vitro. Among those, pigmented epithelialcells were described, resembling retinal pigment epithelium. Preliminaryexperiments carried out at Advanced Cell Technology with primate andhuman ES cell lines show that in a specialized culture system thesecells differentiate into RPE-like cells that can be isolated andpassaged. Human and mouse NT, Cyno parthenote ES cell derivatives havemultiple features of RPE: these pigmented epithelial cells express fourmolecular markers of RPE-bestrophin, CRALBP, PEDF, and RPE65; like RPE,their proliferation in culture is accompanied by dedifferentiation—lossof pigment and epithelial morphology, both of which are restored afterthe cells form a monolayer and become quiescent. Such RPE-like cells canbe easily passaged, frozen and thawed, thus allowing their expansion.Histological analysis of differentiating ES cultures shows a pattern ofcells consistent with early retinal development, including aggregates ofcells similar to rods and cones.

RPE Transplantation

At present, chronic, slow rejection of the RPE allografts preventsscientists from determining the therapeutic efficacy of this RPEtransplantation. Several methods are being considered to overcome thisobstacle. The easiest way is to use systemic immunosuppression, which isassociated with serious side-effects such as cancer and infection. Asecond approach is to transplant the patient's own RPE, i.e. homografts,but this has the drawback of using old, diseased RPE to replace evenmore diseased RPE. Yet, a third approach is to use iris epithelium (IPE)from the same patient but this has the drawback that IPE may not performall the vision related functions of RPE.

The present invention substantially reduces the possibility thattransplantation rejection will occur, because RPE or RPE-like cellsderived from hES cells could be derived from a bank of hES cells withhomozygosity in the HLA region or could be derived from cloned hES celllines. Also, nuclear transfer and parthenogenesis facilitatehistocompatibility of grafted RPE cells and progenitors.

RPE Defects in Retinitis Pigmentosa

Retinitis pigmentosa is a hereditary condition in which the visionreceptors are gradually destroyed through abnormal genetic programming.Some forms cause total blindness at relatively young ages, where otherforms demonstrate characteristic “bone spicule” retinal changes withlittle vision destruction. This disease affects some 1.5 million peopleworldwide. Two gene defects that cause autosomal recessive RP have beenfound in genes expressed exclusively in RPE: one is due to an RPEprotein involved in vitamin A metabolism (cis retinaldehyde bindingprotein), a second involves another protein unique to RPE, RPE65. Withthe use of hES cell derived RPE cell lines cultured without the use ofnon-human animal cells, both of these forms of RP should be treatableimmediately by RPE transplantation. This treatment was inconceivable afew years ago when RP was a hopelessly untreatable and a poorlyunderstood form of blindness.

New research in RPE transplantation suggests there is promise for thetreatment of retinal degeneration, including macular degeneration. Inaddition, a number of patients with advanced RP have regained someuseful vision following fetal retinal cell transplant. One of thepatients, for instance, improved from barely seeing light to being ableto count fingers held at a distance of about six feet from the patient'sface. In a second case, vision improved to ability to see lettersthrough tunnel vision. The transplants in these studies were performedby injection, introducing the new retinal cells underneath the existingneural retina. Not all of the cells survived since the transplantedfetal cells were allogeneic (i.e. not genetically-matched), althoughthose that did survive formed connections with other neurons and beginto function like the photoreceptors around them. Approximately a yearafter the first eight people received the transplants, four haverecovered some visual function and a fifth shows signs of doing so.

Three newly derived human embryonic stem cell lines are similar inproperties to those described earlier (Thomson et al. 1998, Reibunoff etal., 2000, Richards et al., 2000, Lanzendorf et al., 2001): theymaintain undifferentiated phenotype and express known markers ofundifferentiated hES cells, Oct-4, alkaline phosphatase, SSEA-3, SSEA-4,TRA-1-60, TRA-1-81 through 45 passages in culture or over 130 populationdoublings. All hES cell lines differentiate into derivatives of threegerm layers in EB or long term adherent cultures and in teratomas. Oneof the differentiation derivatives of hES cells is similar to retinalpigment epithelium by the following criteria: morphologically, they havea typical epithelial cobblestone monolayer appearance and contain darkbrown pigment in their cytoplasm, which is known to be present in thehuman body only in melanocytes, keratinocytes, retinal and iris pigmentepithelium (IPE). Melanocytes, however, are non-epithelial cells, andkeratinocytes don't secrete but only accumulate melanin. The set ofRPE-specific proteins—bestrophin, CRALBP, PEDF—present in these cellsindicates that they are likely to be similar to RPE and not IPE. Anothersimilarity is the behavior of isolated pigmented cells in culture, whenlittle or no pigment was seen in proliferating cells but was retained intightly packed epithelial islands or re-expressed in newly establishedcobblestone monolayer after the cells became quiescent. Such behaviorwas described for RPE cells in culture (reviewed by Zhao et al., 1997),and it was previously reported (Vinores et al., 1995) that a neuronalmarker tubulin beta III was specifically localized in dedifferentiatingRPE cells in vitro and not in the cells with the typical RPE morphologysuggesting that it reflects the plasticity of RPE and its ability todedifferentiate to a neural lineage. The inventors have observed thesame pattern of tubulin beta III localization in primary and passagedcultures of RPE and RPE-like cells which can reflect a dedifferentiationof such cells in culture or indicate a separate population of cellscommitted to a neuronal fate, that were originally located next topigmented cells through differentiation of hES cells in long-termcultures and could have been co-isolated with RPE-like cells.

In the growing optic vesicle RPE and the neural retina share the samebipotential neuroepithelial progenitor, and their fate was shown to bedetermined by Pax2, Pax6, and Mitf (Baumer et al., 2003), the latterbeing a target of the first two. Pax6 at earlier stages acts as anactivator of proneural genes and is downregulated in the RPE in furtherdevelopment, remaining in amacrine and ganglion cells in mature retina(reviewed by Ashery-Padan and Gruss, 2001). In goldfish, it is alsofound in mitotically active progenitors of regenerating neurons(Hitchcock et al., 1996). The inventors have found that many of theRPE-like cells expressed mitf and Pax6 in a pattern similar to tubulinbeta III and were found only in non-pigmented cells of non-epithelialmorphology that surround pigmented epithelial islands in long termcultures or in cells with a “partial” RPE phenotype (lightly pigmentedand loosely packed). In proliferating cells in recently passagedcultures all these markers were found nearly in every cell suggestingeither a reversal of RPE-like cells to progenitor stage at the onset ofproliferation or massive proliferation of retinal progenitors.Interestingly, in teratomas where islands of pigmented cells ofepithelial morphology were also found, Pax6 was expressed innon-pigmented cells adjacent to pigmented regions (data not shown).Multiple studies have previously shown dedifferentiation of RPE inculture and their transdifferentiation into cells of neuronal phenotype(Reh and Gretton, 1987, Skaguchi et al., 1997, Vinores et al., 1995,Chen et al., 2003), neuronal, amacrine and photoreceptor cells (Zhao etal., 1995), glia (Skaguchi et al., 1997), neural retina (Galy et al.,2002), and to neuronal progenitors (Opaz and Dziak, 1993). Suchprogenitors can in turn coexist with mature RPE-like cells in culture orappear as a result of dedifferentiation of RPE-like cells. At the sametime, cells of neural retina can transdifferentiate into RPE in vitro(Opas et al., 2001), so alternatively, tubulin beta III and Pax6positive cells could represent a transient stage of suchtransdifferentiation of co-isolated neural cells or neural progenitorsinto RPE-like cells.

Differentiation of hES cells into RPE-like cells happened spontaneouslywhen using methods described in the Examples below, and the inventorsnoticed that pigmented epithelial cells reliably appeared in culturesolder than 6-8 weeks and their number progressed overtime—in 3-5 monthscultures nearly every EB had a large pigmented region. In addition tothe described hES lines, six more newly derived hES lines turned intoRPE-like cells, which suggests that since neural fate is usually chosenby ES cells spontaneously, RPE-like cells can arise by default as anadvanced stage of such pathway. It is also possible that in such longterm cultures, where differentiating hES cells form a multi-layeredenvironment, permissive and/or instructive differentiation signals comefrom extracellular matrix and growth factors produced by differentiatingderivatives of hES cells. The model of differentiation of hES cells intoRPE-like cells could be a useful tool to study how such microenvironmentorchestrates RPE differentiation and transdifferentiation.

RPE plays an important role in photoreceptor maintenance, and variousRPE malfunctions in vivo are associated with a number of vision-alteringailments, such as RPE detachment, displasia, atrophy, retinopathy,retinitis pigmentosa, macular dystrophy or degeneration, includingage-related macular degeneration, which can result in photoreceptordamage and blindness. Because of its wound healing abilities, RPE hasbeen extensively studied in application to transplantation therapy. Ithas been shown in several animal models and in humans (Gouras et al.,2002, Stanga et al., 2002, Binder et al., 2002, Schraermeyer et al.,2001, reviewed by Lund et al., 2001) that RPE transplantation has a goodpotential of vision restoration. Recently another prospective niche forRPE transplantation was proposed and even reached the phase of clinicaltrials: since these cells secrete dopamine, they could be used fortreatment of Parkinson disease (Subramanian, 2001). However, even in animmune-privileged eye, there is a problem of graft rejection, hinderingthe progress of this approach if allogenic transplant is used. The otherproblem is the reliance on fetal tissue, as adult RPE has a very lowproliferative potential. The present invention decreases the likelihoodthat graft rejection will occur and removes the reliance on the use offetal tissue.

As a source of immune compatible tissues, hES cells hold a promise fortransplantation therapy, as the problem of immune rejection can beovercome with nuclear transfer technology. The use of the newdifferentiation derivatives of human ES cells, including retinal pigmentepithelium-like cells and neuronal precursor cells, and the use of thedifferentiation system for producing the same offers an attractivepotential supply of RPE and neuronal precursor cells fortransplantation.

EXAMPLES Example 1 Spontaneous Differentiation into Pigmented EpithelialCells in Long Term Cultures

When hES cell cultures are allowed to overgrow on MEF in the absence ofLIF, FGF and Plasmanate, they form a thick multilayer of cells. About 6weeks later, dark islands of cells appear within the larger clusters(FIG. 1). These dark cells are easily seen with the naked eye and lookedlike “freckles” in a plate of cells as shown in FIG. 1A. At highermagnification these islands appear as tightly packed polygonal cells ina cobblestone monolayer, typical of epithelial cells, with brown pigmentin the cytoplasm (FIG. 1C). There are differences in the amount ofpigment in the cells with cells in the central part of the islandshaving the most pigment and those near the edges the least. (FIGS. 1Eand 1F).

When hES cells form embryoid bodies (EB)—pigmented epithelial cellsappear in about 1-2% of EBs in the first 6-8 weeks (FIG. 1B). Over timemore and more EBs develop pigmented cells, and by 3 months nearly everyEB had a pigmented epithelial region (FIG. 1D). Morphology of the cellsin the pigmented regions of EBs was very similar to that of adherentcultures (FIG. 1D).

Example 2 Isolation and Culture of Pigmented Epithelial Cells

The inventors isolated pigmented epithelial cells from both adherent hEScell cultures and from EBs. Pigmented polygonal cells were digested withenzymes (trypsin, and/or collagenase, and/or dispase), and the cellsfrom these pigmented islands were selectively picked with a glasscapillary. Although care was taken to pick only pigmented cells, thepopulation of isolated cells invariably contained some non-pigmentedcells. After plating cells on gelatin or laminin for 1-2 days, the cellswere considered to be primary cultures (P0).

Primary cultures contained islands of pigmented polygonal cells as wellas some single pigmented cells. After 3-4 days in culture, non-pigmentedcells that seemed to have lost epithelial morphology (flatter and cellswith lamellipodia) appeared at the periphery of some islands (FIG. 2).The number of such peripheral cells increased over time, suggesting thatthese cells were proliferating, and after 2 weeks most cells in thenewly formed monolayer contained very little or no pigment. Aftercontinued culture, for another 2-3 weeks, pigmented epithelial cellsbegan to reappear, visibly indistinguishable from those in the originalcultures (FIG. 2).

Example 3 Detection of RPE Markers

The preliminary characterization of these differentiated human cells asRPE is based on their similarity to RPE cultures previously described;principally, their epithelial morphology and possession of pigment.There are three types of pigmented epithelial cells in human body:retinal and iris pigmented epithelium and keratinocytes, but the latterdon't secrete pigment. The epithelial structure and cobblestonemorphology are not shared by other pigmented cells, e.g. melanocytes. Itis also noteworthy that RPE cells have been shown to lose and regaintheir pigment and epithelial morphology when grown in culture (Zhao1997, Opas and Dziak, 1994), and the pigmented cells behaved in asimilar manner, so to test the hypothesis that the ES derived cells maybe RPE, they were stained with antibodies to known markers for RPE:bestrophin and CRALBP. FIG. 3 (left panel) shows membrane localizationof bestrophin (A) and CRALBP (C), both are found in pigmented epithelialislands. Not all of the cells stain with these antibodies and intensityof staining correlated with pigment expression and “tightness” ofcolonies—the borders of each pigmented island where cells were largerand more loosely packed showed lower expression of both proteins.

To further characterize presumably RPE cells, analysis was performed onthe expression of bestrophin, CRALBP by Western blotting. FIG. 3 (rightpanel, top) shows the bands, corresponding to bestrophin, 68 kD (a),CRALBP, 36 kD (b) in cell lysates. All these proteins were found in bothprimary cultures and subsequent passages.

Another known PRE marker, RPE65, was found in the RPE-like cells byreal-time RT-PCR (FIG. 3, right panel, bottom). As shown in FIG. 3,right panel, bottom, expression of RPE65 was confirmed in all HES-RPEsamples analyzed. Interestingly, mature cultures (seven weeks afterpassaging) had four to nine folds more RPE65 mRNA than the controlundifferentiated hES cells, whereas earlier passage (two-week old)cultures only exceeded the control by 1.5 to 2.5 fold. See FIG. 3, rightpanel, bottom.

PEDF ELISA assay showed the presence of PEDF in cell lysates of allpresumed RPE cultures, and Western blot showed a band of approximately48 kD (not shown).

Detection of Markers of Neuronal and Retinal Progenitors in RPE-LikeCultures

PAX-6, Pax2, mitf and tubulin beta III were shown to be expressed in themajority of cells in recently passaged and only in a small number ofcells in old cultures of RPE-like cells derived from hES cells (FIG. 4).

In proliferating cultures (day 3 after trypsinization) where RPE-likemorphology of the proliferating cells is lost, nearly every cell showedthe presence of mitf, Pax6, tubulin beta III and nestin. Pax2 was foundonly a small subset of cells which appeared mitf-negative, while therewas a strong degree of co-localization of Pax6/mitf, mitf/tubulin betaIII, and Pax6/tubulin beta III. In 21 days old quiescent cultures afterpigmented epithelial islands were reestablished, groups of PAX-6 andmitf were found mostly in non-pigmented cells of non-epithelialmorphology between pigmented epithelial islands (FIG. 4, A-C). andtubulin beta III had a similar pattern of distribution (not shown).However, there were populations of mitf-positive and Pax6-negativecells, located close to the periphery of pigmented islands (FIG. 4,A-C). Pax2 was found only in a very small subset of mitf-negative cells(FIG. 4, E-H). No presence of either of these proteins was ever detectedin the cells of “mature” pigmented epithelial islands. However, thesemarkers in cells that only had some RPE features were often visible,i.e. either looked epithelial but had no pigment or in certain singlepigmented cells away from pigmented epithelial islands.

Example 4 Characterization of RPE-Like Cells Derived from hES Cell LineAct J-1 from Cyno-1 ES Cells and Derivation of RPE-Like Cells fromExisting HES Cell Lines H1, H9, and H7

An RPE-like cell line is expanded, tested for freezing and recovery, andcharacterized using the following methods and molecular markers of RPEcells: bestrophin and CRALBP by Western blot and immunofluorescence,PEDF by ELISA and Western blot, and RPE65 by RT-PCR. The cells areinjected in SCID mice with undifferentiated hES or Cyno-1 cells as acontrol to evaluate tumorigenicity. Karyotyping of RPE-like cells willbe done by a clinical laboratory on a commercial basis. Characterizationof the functional properties of RPE-like cells and studies of theirtransplantation potential are then carried out as otherwise described inthis application and also using those techniques known to those skilledin the art.

Gene expression profiling experiments are done using Affymetrix humangenome arrays. Gene expression is compared in RPE-like cells derivedfrom ES cells and in retinal samples from autopsies. Several animalmodels can be used to verify the effectiveness of the transplantedRPE-like cells, including but not limited to, rhesus monkey, rat, andrabbit.

Example 5 Optimization of the Differentiation Culture System EnsuringHigh Yields of RPE-Like Cells

ES cells are cultured on feeder cells or as embryoid bodies (EB) in thepresence of factors such as bFGF, insulin, TGF-beta, IBMX, bmp-2, bmp-4or their combinations, including stepwise addition. Alternatively, EScells are grown on various extracellular matrix-coated plates (laminin,fibronectin, collagen I, collagen IV, Matrigel, etc.) in evaluating therole of ECM in RPE formation. Expression of molecular markers of earlyRPE progenitors (Pax6, Pax2, mitf) and of RPE cells (CRALBP, bestrophin,PEDF, RPE65) are evaluated at various time intervals by real-time RT-PCRto verify and determine successful combinations of the above mentionedagents and stepwise procedure that produces enrichment in RPE-like cellsor their progenitors. This approach can also be used to produce commonprogenitors of RPE and other eye tissues, such as photoreceptor orneural retina which can be isolated and further characterized for theirdifferentiation potential and used in transplantation studies.

Example 6 Derivation of RPE and Other Eye Tissue Progenitors FromExisting and New ES Cell Lines

Using the data from the gene expression profiling, expression of the RPEprogenitor markers will be correlated with the expression of the surfaceproteins in order to find a unique combination of surface markers forRPE progenitor cells. If such markers are found, antibodies to surfaceproteins can be used to isolate a pure population of RPE progenitorsthat can be then cultured and further differentiated in culture or usedin transplantation studies to allow their differentiation aftergrafting.

If the data from the gene expression profiling experiments isinsufficient, to isolate the RPE progenitors the following approach willbe used. ES cells and RPE-like cells will be transfected with GFP underthe control of a promoter such as Pax6, and stable transfectants will beselected. From a culture of transfected differentiating ES cells orproliferating (dedifferentiated) RPE cells, GFP/Pax6-positive cells willbe isolated by FACS and used as an antigen source for mouse injection toraise monoclonal antibodies to the surface molecules of Pax6 positivecells. Because Pax6 is present not only in RPE progenitors, screeningwill be done (by FACS) using several strategies: a) againstproliferating RPE-like cells, b) against Pax2-positive RPE cells, c)against mitf-positive RPE cells. For b) and c) RPE cells will betransfected with GFP under the corresponding promoter; as a negativecontrol, RPE or ES cells negative by these antigens will be used. Afterexpansion of positive clones selected by all three strategies,antibodies will be tested against all types of cells used in screeningand further analyzed: since this strategy can produce antibodies thatrecognize cell surface antigens specific and non-specific for RPEprogenitors, the cells from differentiating total population of ES cellsor of RPE cells selected with these antibodies will be assessed formolecular markers of RPE progenitors and for their ability to produceRPE.

Using the optimized defined stepwise procedures to produce RPE or otherearly progenitors of eye tissues and the antibodies to their uniquesurface markers, such progenitors will be isolated from differentiatedES cells and cultured in vitro. Their ability to differentiate intovarious tissues of the eye will be investigated using the strategydescribed in Aim 2.

ES cell lines that already produced RPE-like cells (H1, H7, H9, ACT J-1,ACT-4, Cyno-1), RPE-like cells will be used to continue to deriveRPE-like cells and their progenitors as described in Aims 1 and 2. Afterexpansion and characterization for molecular markers of RPE, these lineswill be single-cell cloned, and the resulting lines will becharacterized as described in Aim 1. The lines meeting criteria for RPEcells will be used for transplantation studies. New human ES cell lineswill be derived from unused IVF embryos, from donated oocytes,stimulated to develop without fertilization (parthenote), and fromgenerated developing blastocysts obtained from donated oocytes with theapplication of nuclear transfer technology. RPE-like cells and commoneye progenitors will be derived from these lines using the approach inAim 2, and the resulting lines will be characterized as in Aim 1.[Optional] new human ES cell lines will be derived in a virus-freesystem, characterized and submitted for clinical trials.

Example 7 Therapeutic Potential of RPE-Like Cells and Progenitors inVarious Animal Models of Retinitis Pigmentosa & Macular Degeneration

Primate ES cells are tested in cynomologus monkeys (Macaques).Initially, vitrectomy surgery is performed and the cells aretransplanted into the subretinal space of the animals. The first step isthe transplantation of the cells in the suspension format after which asubstrate or matrix is used to produce a monolayer transplantation. Thiscan also be performed in immunosuppressed rabbits using cells derivedfrom human ES-cells and also in various other animal models of retinitispigmentosa, including rodents (rd mouse, RPE-65 knockout mouse,tubby-like mouse, RCS rat, cats (Abyssinian cat), and dogs (conedegeneration “cd” dog, progressive rod-cone degeneration “prcd” dog,early retinal degeneration “erd” dog, rod-cone dysplasia 1, 2 & 3 “rcd1,rcd2 & rcd3” dogs, photoreceptor dysplasia “pd” dog, and Briard “RPE-65)dog). Evaluation is performed using fluorescent angiography, histology(whether or not there is photoreceptor restoration and possibly ERG.Functional testing will also be carried out, including phagocytosis(photoreceptor fragments), vitamin A metabolism, tight junctionsconductivity, and electron microscopy.

Example 8 Direct Differentiation of RPE Cells From Human Embryo-DerivedCells

Human blastocyst-staged embryos are plated in the presence of murine orchick embryo fibroblasts with or without immunosurgery to remove thetrophectoderm or directly plates on extracellular matrix protein-coatedtissue cultureware. Instead of culturing and passaging the cells toproduce a human ES cell line, the cells are directly differentiated.

When hEDC cell cultures are allowed to overgrow on MEF in the absence ofLIF, FGF and Plasmanate, they will form a thick multilayer of cells.(Alternate growth factors, media, and FBS can be used to alternatedirect differentiation as is known to those skilled in the art.) About 6weeks later, dark islands of cells will appear within the largerclusters. These dark cells are easily seen with the naked eye and lookedlike “freckles” in a plate of cells as shown in FIG. 5B. At highermagnification these islands appear as tightly packed polygonal cells ina cobblestone monolayer, typical of epithelial cells, with brown pigmentin the cytoplasm (FIG. 5A). There are differences in the amount ofpigment in the cells with cells in the central part of the islandshaving the most pigment and those near the edges the least. (FIG. 5B).

When hEDC cells are directly differentiated they may, though typicallyhave not, formed embryoid bodies (EB). Pigmented epithelial cells appearin about 1-2% of these differentiated cells and/or EBs in the first 6-8weeks. Over time more and more EBs develop pigmented cells; and by 3months nearly every EB had a pigmented epithelial region. Morphology ofthe cells in the pigmented regions of EBs was very similar to that ofadherent cultures.

Materials and Methods:

MEF medium: high glucose DMEM, supplemented with 2 mM GlutaMAX I, and500 u/ml Penicillin, 500 ug/ml streptomycin (all from Invitrogen) and16% FCS (HyCLone). hES Cells Growth medium: knockout high glucose DMEMsupplemented with 500 u/ml Penicillin, 500 μg/ml streptomycin, 1%non-essential amino acids solution, 2 mM GlutaMAX I, 0.1 mMbeta-mercaptoethanol, 4 ng/ml bFGF (Invitrogen), 1-ng/ml human LIF(Chemicon, Temecula, Calif.), 8.4% of Serum Replacement (SR, Invitrogen)and 8.4% Plasmanate (Bayer). Derivation medium contained the samecomponents as growth medium except that it had lower concentration of SRand Plasmanate (4.2% each) and 8.4% FCS and 2× concentration of humanLIF and bFGF, as compared to growth medium. EB medium: same as growthmedium except bFGF, LIF, and Plasmanate; the SR concentration was 13%.RPE medium: 50% EB medium and 50% MEF medium.

hES Cell Lines

Differentiation experiments were performed with adherent hES cells orwith embryoid bodies (EBs). For adherent differentiation, hES cells wereallowed to overgrow on MEFs until the hES colonies lost their tightborders at which time the culture media was replaced with EB medium(usually, 8-10 days after passaging). The medium was changed every 1-2days. For EB formation, hES cells were trypsinized and cultured in EBmedium on low adherent plates (Costar).

Immunostaining

Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% NP-40for localization of intracellular antigens, and blocked with 10% goatserum, 10% donkey serum (Jackson Immunoresearch Laboratories, WestGrove, Pa.) in PBS (Invitrogen) for at least one hour. Incubation withprimary antibodies was carried out overnight at 4° C., the secondaryantibodies (Jackson Immunoresearch Laboratories, West Grove, Pa.) wereadded for one hour. Between all incubations specimens were washed with0.1% Tween-20 (Sigma) in PBS 3-5 times, 10-15 minutes each wash.Specimens were mounted using Vectashield with DAPI (Vector Laboratories,Burlingame, Calif.) and observed under fluorescent microscope (Nikon).Antibodies used: bestrophin (Novus Biologicals, Littleton, Colo.),anti-CRALBP antibody was a generous gift from Dr. Saari, University ofWashington. Secondary antibodies were from Jackson ImmunoresearchLaboratories, and Streptavidin-FITC was purchased from Amersham.

Isolation and Passaging of RPE-Like Cells

Adherent cultures of hES cells or EBs were rinsed with PBS twice andincubated in 0.25% Trypsin/1 mM EDTA (Invitrogen) at 37° C. until themonolayer loosened. Cells from the pigmented regions were scraped offwith a glass capillary, transferred to MEF medium, centrifuged at 200×g,and plated onto gelatin-coated plates in RPE medium. The medium waschanged after the cells attached (usually in 1-2 days) and every 5-7days after that; the cells were passaged every 2-4 weeks with 0.05%Trypsin/0.53 mM EDTA (Invitrogen).

Western Blot and ELISA

Samples were prepared in Laemmli buffer (Laemmli, 1970), supplementedwith 5% Mercaptoethanol and Protease Inhibitor Cocktail (Roche), boiledfor 5 minutes and loaded onto a 8-16% gradient gel (Bio-Rad, Hercules,Calif.) using a Mini-Protean apparatus; the gels were run at 25-30 mAper gel; proteins were transferred to a 0.2 Nitrocellulose membrane(Schleicher and Shull, Keene, N.H.) at 20 volt overnight. Blots werebriefly stained with Ponceau Red (Sigma) to visualize the bands, washedwith Milli-Q water, and blocked for 1 hour with 5% non-fat dry milk in0.1% TBST (Bio-Rad). Primary antibodies to bestrophin, CRALBP or PEDF(Chemicon) were added for 2 hours followed by three 15-minute washeswith TBST; peroxidase-conjugated secondary antibodies were added for 1hour, and the washes were repeated. Blots were detected using ECL systemwith Super-Signal reagent (Pierce). PEDF ELISA was performed on celllysates using PEDF ELISA kit (Chemicon) according to manufacturer'sprotocol.

Real-Time RT-PCR

Total RNA was purified from differentiating ES cultures by a two-stepprocedure Crude RNA was isolated using Trizol reagent (Invitrogen) andfurther purified on RNeasy minicolumns (Qiagen). The levels of RPE65transcripts were monitored by real-time PCR using a commercial primerset for RPE65 detection (Assay on Demand # Hs00165642_m1, AppliedBiosystems) and Quantitect Probe RT-PCR reagents (Qiagen), according tothe manufacturer's (Qiagen) protocol.

Example 9 Use of Transcriptomics to Identify Normal Differentiated CellsDifferentiated Ex Vivo

hES-cell derivatives are likely to play an important role in the futureof regenerative medicine. Qualitative assessment of these and other stemcell derivatives remains a challenge that could be approached usingfunctional genomics. We compared the transcriptional profile of hES-RPEvs. its in vivo counterpart, fetal RPE cells, which have beenextensively researched for its transplantation value. Both profiles werethen compared with previously published (Rogojina et al., 2003)transcriptomics data on human RPE cell lines.

The gene expression profile of our data set was compared to two humanRPE cell lines (non-transformed ARPE-19 and transformed D407, Rogojinaet al., 2003) to determine whether HES-RPE have similar globaltranscriptional profiles. To account for common housekeeping genesexpressed in all cells, we used publicly available Affymetrix data setsfrom undifferentiated hES cells (H1 line, h1-hES,—Sato et al., 2003) andbronchial epithelial cells (BE, Wright et al., 2004) as a control basedon its common epithelial origin that would allow to exclude commonhousekeeping and epithelial genes and identify RPE-specific genes.

There were similarities and differences between HES-RPE, hES-RPE-TD,ARPE-19, D407. The similarities were further demonstrated by analyzingthe exclusive intersection between those genes present inhES-RPE/ARPE-19 but not in BE (1026 genes). To account for background,we compared this to the exclusive intersection of genes present inBE/hES-RPE, but not ARPE-19 (186 genes), which results in a five- tosix-fold greater similarity in HES-RPE and ARPE-19 when compared to BE.D407/ARPE19 appear to lose RPE specific genes such as RPE65, Bestrophin,CRALBP, PEDF, which is typical of long-term passaged cells (FIG. 6).Further data mining revealed known RPE specific ontologies such asmelanin biosynthesis, vision, retinol-binding, only in fetal RPE andES-RPE but not ARPE19.

Comparison of hES-RPE, ARPE-19 and D407 to their in vivo counterpart,freshly isolated human fetal RPE (feRPE), was in concordance with ourprevious data, demonstrating that the transcriptional identity ofHES-RPE to human feRPE is significantly greater than D407 to fe RPE (2.3fold difference-849 genes/373 genes) and ARPE-19 to feRPE (1.6 folddifference-588 genes/364 genes (FIGS. 5 c/5 d). The RPE specific markersidentified above, which were only present in HES-RPE and not in ARPE-19or D407 were also present in feRPE, demonstrating a higher similarity ofhES-RPE to its in vivo counterpart than of the cultured RPE lines.

Seven-hundred-and-eighty-four genes present in HES-RPE were absent infeRPE and ARPE-19 data sets. Since the retention of “stemness” genescould potentially cause transformation of hES derivatives into malignantteratomas if transplanted into patients, we created a conservativepotential “stemness” genes data using currently available Affymetrixmicroarray data sets Abeyta et. al 2004 Sato 2003). This resulted in alist of 3806 genes present in all 12 data sets (including commonhousekeeping genes). j Only 36 of the 784 genes present in the HES-RPEdata set but not feRPE-ARPE-19 were common to the 3806 potentialsternness genes. None of these were known sternness genes such as Oct4,Sox2, TDGF1.

Example 10 Use of RPE Cells for Treatment of Parkinson's Disease

hRPE can be used as an alternative source of cells for cell therapy ofParkinson's Disease because they secrete L-DOPA. Studies have showedthat such cells attached to gelatin-coated microcarriers can besuccessfully transplanted in hemiparkinsonian monkeys and producednotable improvements (10-50) thousand cells per target), and inFDA-approved trial started in 2000 the patients received hRPEintrastriatial transplants without adverse effects. One of the manyadvantages to the use of hES cell-derived RPE is that it circumvents theshortage of donor eye tissue. It also facilitates the use of genetherapy.

Example 11 Use of Stem Cell Derived RPE Cell Line for Rescuing orPreventing Photoreceptor Loss

Derivation of RPE Cell Lines

Human embryonic stem (“hES”) cells were grown in MEF medium containinghigh glucose DMEM, supplemented with 2 mM GlutaMAX I or glutamine, 500u/ml Penicillin, 500 μg/ml streptomycin (all from Invitrogen) and 16%FCS (can range from 8 to 20%) (HyCLone). hES cells may be grown ingrowth medium containing knockout high glucose DMEM supplemented with500 u/ml Penicillin, 500 μg/ml streptomycin, 1% non-essential aminoacids solution, 2 mM GlutaMAX I, 0.1 mM beta-mercaptoethanol, 4 ng/ml(or up to 80) bFGF (Invitrogen), 10 ng/ml (or up to 100) human LIF (LIFis optional) (Chemicon, Temecula, Calif.), 8.4% of Serum Replacement(can be used up to 20%) (SR, Invitrogen) and 8.4% Plasmanate (optional)(Bayer). EB medium is the same as growth medium except bFGF, LIF, andPlasmanate are not included and the SR concentration was 13%. RPE mediumis 50% EB medium and 50% MEF medium. Alternatively, hES cells can becultured in the presence of human serum or FBS. For RPE culture,different media can be used that supports its proliferation,transdifferentiation and re-establishment of differentiated phenotype.Examples include, but are not limited to, high glucose DMEM supplementedwith 2 mM GlutaMAX I or glutamine, 500 u/ml Penicillin, 500 μg/mlstreptomycin (antibiotics are optional) (all from Invitrogen) and 16%FCS (can range from 8 to 20%) (HyClone) or human serum; 1:1 mixture ofDulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES 0.5 mM sodiumpyruvate, fetal bovine serum, 10%; (from ATCC, recommended forpropagation of ARPE-19 cell line established from human RPE cells). Acell culture medium that supports the differentiation of human retinalpigment epithelium into functionally polarized monolayers may also beemployed for this purpose.

RPE may be cultured as previously described (Hu and Bok, MolecularVision (2000) 7:14-19, the disclosure of which is incorporated byreference) or other culture medium which has serum or serum replacementcomponents or growth factor combination that supports RPE growth.

Two of the hES cell used for these studies were derived as described(Cowan et al., N. Eng. J. Med. 350: 1353-1356 (2004), Klimanskaya andMcMahon, Handbook of Stem Cells, Vol. 1: Embryonic Stem Cells, Edited byLanza et al., Elsevier/Academic Press, pp. 437-449 (2004), thedisclosures of both are incorporated by reference), three lines werederived by Jamie Thomson (H1, H7, & H9) Human frozen blastocysts weredonated to the study by couples who had completed their fertilitytreatment.

Differentiation experiments were performed with adherent hES cells orwith embryoid bodies (EBs). For adherent differentiation, hES cells wereallowed to overgrow on MEFs until the hES colonies lost their tightborders, at which time the culture media was replaced with EB medium(usually, 8-10 days after passaging). The medium was changed as itbecame yellow or every 1-2 days for dense cultures and less frequentlyfor sparse cultures or EBs. For EB formation, hES cells were trypsinizedand cultured in EB medium on low adherent plates (Costar).

Immunostaining

Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% NP-40for localization of intracellular antigens, and blocked with 10% goatserum, 10% donkey serum (Jackson Immunoresearch Laboratories, WestGrove, Pa.) in PBS (Invitrogen) for at least one hour. The specimen werethen incubated with primary antibodies overnight at 4° C., and thenincubated with secondary antibodies (Jackson ImmunoresearchLaboratories, West Grove, Pa.) for one hour. Between all incubations,the specimens were washed with 0.1% Tween-20 (Sigma) in PBS 3-5 timesfor 10-15 minutes each wash. Specimens were mounted using Vectashieldwith DAPI (Vector Laboratories, Burlingame, Calif.) and observed underfluorescent microscope (Nikon). Antibodies used include anti-bestrophinantibody (Novus Biologicals, Littleton, Colo.), and anti-CRALBP antibody(a gift from Dr. Saari, University of Washington). Secondary antibodieswere obtained from Jackson Immunoresearch Laboratories, andStreptavidin-FITC was purchased from Amersham.

Isolation and Passaging of RPE-Like Cells

Adherent cultures of hES cells or EBs were rinsed with PBS twice andincubated in 0.25% Trypsin/1 mM EDTA (Invitrogen) at 37° C. until themonolayer loosened. Cells from the pigmented regions were scraped offwith a glass capillary, transferred to MEF medium, centrifuged at 200×g,and plated onto gelatin-coated plates in RPE medium. The medium waschanged after the cells attached (usually in 1-2 days) and every 5-7days after that. The cells were passaged every 2-4 weeks with 0.05%Trypsin/0.53 mM EDTA (Invitrogen).

Cells may be passaged or collected for transplantation using trypsin orcollagenase IV, collagenase I, or dispase at concentrations of 1-10%.Any combination of these at concentrations of 1-10% each could be usedinstead of trypsin for isolation and passaging of pigmented cells.“Combination” in this context is intended to mean use of such enzymestogether or sequentially—e.g., collagenase digestion followed bytrypsin. The passage dilution may vary from no dilution to 1:6 orhigher. The substrate for culture prior to transplantation may beanything that supports growth and features of hES-RPE, such as, but notlimited to, gelatin, fibronectin, laminin, collagen or different typesof extracellular matrix, uncoated plastic surface, filters—uncoated orcoated with ECM (extracellular matrix) proteins, Matrigel, ECM isolatedfrom other cell cultures, such as cornea, RPE, fibroblasts, uncoatedbeads, or beads coated with ECM. The time between passaging can varyfrom one day to several weeks. In prior experiments, nine-month oldembryoid bodies with sheets of RPE on the surface were used to establishpassagable cultures of hES-RPE so there is no limit known on how longthe cells can be kept in culture without passaging.

Cultures may consist not only of cells with proper RPE morphology—i.e.polygonal tightly packed pigmented cells—but also of cells with varyingdegree of transdifferentiation (elongated pigmented or non-pigmentedcells, etc.) and other cell types that co-differentiate from hES cells.Unless cells are individually selected for culturing, such culturesusually contain RPE islands that are separated by non-RPE cells.

The cultures of differentiating ES cells that exhibited the signs ofdifferentiation along the neural lineage (expressing markers of thislineage, such as nestin, Pax6, etc., as could be detected by RT-PCR,Western blot, immunostaining, histology, or morphology of the individualcells which could be islands of pigmented cells, or epithelial sheets,or aggregates of vacuolated cells) were passaged with trypsin,collagenase, dispase, or mixture of such, expanded and cultured untilthe pigmented epithelial islands appeared or multiplied in numbers(usually, one or two passages). Such mixed cultures of pigmentedepithelial and non-pigmented non-epithelial cells could be used toselectively hand-pick pigmented and non-pigmented cells aftercollagenase or collagenase-dispase digestion. These hand-pickedpigmented and non-pigmented cells could then be dispersed into smalleraggregates and single cells or plated without dispersion, resulting inestablishment of high purity RPE cultures.

Western Blot and ELISA

Samples were prepared in Laemmli buffer (Laemmli, 1970), supplementedwith 5% Mercaptoethanol and Protease Inhibitor Cocktail (Roche), boiledfor 5 minutes and loaded onto a 8-16% gradient gel (Bio-Rad, Hercules,Calif.) using a Mini-Protean apparatus; the gels were run at 25-30 mAper gel. Proteins were then transferred from the gel to a 0.2Nitrocellulose membrane (Schleicher and Shull, Keene, N.H.) at 20 voltovernight. Blots were briefly stained with Ponceau Red (Sigma) tovisualize the bands, washed with Milli-Q water, and blocked for 1 hourwith 5% non-fat dry milk in 0.1% TBST (Bio-Rad). Primary antibodies tobestrophin, CRALBP or PEDF (Chemicon) were added to the blot for 2 hoursfollowed by three 15-minute washes with TBST. peroxidase-conjugatedsecondary antibodies were then added to the blot for 1 hour, and thewashes were repeated. Blots were detected using ECL system withSuper-Signal reagent (Pierce). PEDF ELISA was performed on cell lysatesusing PEDF ELISA kit (Chemicon) according to the manufacturer'sprotocol.

Real-Time RT-PCR

Total RNA was purified from differentiating ES cultures by a two-stepprocedure. Crude RNA was isolated using Trizol reagent (Invitrogen) andfurther purified on RNeasy minicolumns (Qiagen). The levels of RPE65transcripts were monitored by real-time PCR using a commercial primerset for RPE65 detection (Assay on Demand # Hs00165642_m1, AppliedBiosystems) and QuantiTect Probe RT-PCR reagents (Qiagen), according tothe manufacturer's (Qiagen) protocol.

Transplantation of hES-Derived RPE Cell Line

Cultures of hES cell lines may be used as transplant cells to one eye of23-day old RCS rats to rescue or prevent photoreceptor loss. Cells ofdifferent morphology and/or of different degrees of differentiation maybe chosen. Transplantation may be done as described (Lund et al. (2001)Proc. Natl. Acad. Sci. USA 98: 9942-47; Del Priore et al. InvestigativeOphthalmology & Visual Science (2004) 45: 985-992; Gouras et al. (2002)Ophthalmology & Visual Science 43: 3307-11, the disclosures of which areincorporated by reference herein). Following digestion with trypsin orother enzyme (as described above), hES cells may be washed, anddelivered trans-sclerally in a suspension at a density of 2×10⁵ cellsper 2 μl injection. Delivery may be achieved in Ham's F-10 medium withthe use of a fine glass pipette with internal diameter of about 75-150μm. Injections are to be delivered into the dorso-temporal subretinalspace of one eye of anesthetized 23-day old, dystrophic-pigmented RCSrats, at a time before functional deterioration and significantphotoreceptor death. Sham-injected rats receive carrier medium alonewithout hES-derived cells. Histological assessment of postoperative ratsmay be done at shorter time points (e.g., at 1 month post-operatively)to assess short-term changes associated with transplantation and atlonger time points (e.g., at 5 months post-operatively) to examine donorcell survival. Cells may be tagged or labeled by culturing in mediumcontaining 20 μm BrdUrd for 48 hours before transplantation.

Functional Assessment of Transplanted hES-Derived Cells

Also described in Lund et al. (2001) Proc. Natl. Acad. Sci. USA 98:9942-47, behavioral assessment of grafted rats may be performed with ahead-tracking apparatus that consists of a circular drum rotating at aconstant velocity of 12 degrees/sec around a stationary holding chambercontaining the animal. Presenting stimuli may be placed oninterchangeable panels covered with black and white stripes with varyingspatial frequencies such as 0.12.5, 0.25, and 0.5 cycles per degree.Animals may be tested at 10-20 weeks postoperatively. All animalassessments may be conducted blindly by a sole operator. Behavioral datamay be analyzed using ANOVA.

Physiological studies may be conducted on animals with cornealelectrocardiograms (ERGs) at 60 and 90 days. Prior to testing, the ratsare to be adapted to the dark overnight and anesthetized under red lightwith ketamine and xylazine. See, for example, Peachy et al., VisNeurosci. 2002 November-December; 19(6):693-701. The pupils are to bedilated and ERGs recorded from the cornea with a cotton wick salineelectrode. Subcutaneous 30-gauge needles may be inserted into theforehead and trunk as reference and ground electrodes, respectively.While maintaining the subject rat body temperature at 35-36° C., a lightstimulus is to be applied at a maximum flash intensity measured at thecornea of about 0.7×103 μW/cm 2. Responses are to be recorded andaveraged by a computerized data acquisition system at varyingfrequencies. ERG amplitudes are to be measured from the initial negativepeak of the a-wave or from the baseline to the positive peak of theb-wave.

As in Lund et al. (2001) Proc. Natl. Acad. Sci. USA 98: 9942-47,threshold responses to illumination of visual receptive fields from thesuperior colliculus at 100 days are to be recorded. Animals are to beplaced under terminal urethane anesthesia (1.25 g/kg i.p.). Data is tobe collected over the entire visual field at independent points spacedroughly 200 μm apart, with each point corresponding to about 10-15°displacements in the visual field. Visual thresholds are to be measuredas the increase in intensity over background and maintained at 0.02cd/m² [at least 2.6 logarithm (log) units below rod saturation] foractivating units in the superficial 200 μm of the superior colliculuswith a spot light of 3° diameter. Threshold maps are to be generated foreach animal and illustrated as retinal representations.

Seven eyes transplanted with RPE cells derived from an H9 cell line andsix eyes transplanted with RPE cells derived from a J1 cell line weresubjected to ERG analysis. RPE transplants were conducted with 23 dayold rats, and ERG analysis was done 36 days following transplantation.The H9 group yielded uniformly good responses, while the J1 groupyielded 1 animal with only minimal response.

Histological Analysis of Transplanted Cells

As described in Lund et al. (2001) Proc. Natl. Acad. Sci. USA 98:9942-47, animals are to be subjected to histological analysis. Animalsmay be euthanized with Euthanal and perfused transcardially with PBSfollowing by periodate-lysine-paraformaldehyde (PLP). Eyes are to besectioned and stained with cresyl violet. Eyes from animals of the BrdUgroup are to be labeled with anti-BrdUrd antibody and visualized withthe use of an appropriate secondary antibody and respective reagents.Other eyes may be fixed by injection with 2.5% paraformaldehyde, 2.5%glutaraldehyde, and 0.01% picric acid in 0.1 M cacodylate buffer. Eyesmay be postfixed in 1% osmium tetroxide, and subsequently dehydratedthrough graded alcohol to epoxypropane. Tissue may be embedded in resinfrom which semi-thin sections may be cut and stained with toluidine bluein 1% borate buffer.

Example 12 Use of Stem Cell Derived Neural Progenitor Cells for theTreatment of Retinal Degeneration

Generation of Neural Progenitors

hES cells (e.g. H1, H7 and H9, National Institutes of Health—registeredas WA01, WA07 and WA09) are allowed to overgrow on MEF medium (highglucose DMEM, supplemented with 2 mM GlutaMAX I or glutamine, and 500u/ml penicillin, 500 μg/ml streptomycin (antibiotics optional) (all fromInvitrogen) and 15% FCS (can range from 8% to 20%) (HyClone)) or onextracellular matrix. And after one week or longer after passaging, thehES cells are split with trypsin, collagenase or dispase, or acombination of the two latter enzymes, and plated on gelatin in EBmedium (see Example 11). The medium is changed as it gets yellow,usually every 2-4 days. The majority of the cells growing under theseconditions are positive as neural progenitors because they expressnestin and/or tubulin beta III and have typical appearance of neuralprogenitor cells—elongated spindle-like cells. They can be passagedagain under the same conditions, which leads to enrichment of the cellpopulation with nestin- and tubulin beta III-positive cells. RT-PCR isused to confirm the presence of nestin, Pax6, N-CAM, tubulin beta III insuch cultures.

Alternatively, spheroids forming in differentiating cultures of hEScells can be removed and plated onto cell culture dishes (could becoated with gelatin or another extracellular matrix, permitting theformation of the described cell type) in EB medium, or such spheroidscould form after the first passage of the total population ofdifferentiating hES cells and can be approached in the same way. Withina few days, growth of spindle-like cells is noticed, which can later beexpanded and express the above mentioned markers for neural progenitorcells.

Differentiation of Ocular Tissues from hES Cells

Differentiation conditions as described in Example 11 of hES cells allowfor the appearance of rod and cone-like structures as shown byhistological examination of differentiating cultures (FIG. 9 a) and byRT-PCR analysis, which confirms expression of rhodopsin, opsin 5, opsin1, and recoverin (FIGS. 9 b, 9 c and 9 d). We also show that suchcultures may contain corneal cells, as RT-PCR detected keratin 12, acorneal marker (FIG. 9 e).

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions.

1. A method for producing an enriched population of human retinalpigment epithelium (RPE) cells, the method comprising: (a) providing amultilayer population of human embryonic stem (hES) cells; (b) culturingsaid multilayer population of hES cells under conditions that do notmaintain the undifferentiated state of said hES cells for a sufficienttime to allow for the appearance of putative human RPE cells, whereinsaid putative human RPE cells comprise brown pigment dispersed withintheir cytoplasm; (c) selecting one or more of said putative human RPEcells from the culture of step (b) to obtain human RPE cells; and (d)culturing said human RPE cells obtained_in step (c) to form a cellmonolayer containing cells that are Pax6− and bestrophin+ and exhibit acharacteristic cobblestone, polygonal, epithelial-like appearance andcomprise brown pigment dispersed within their cytoplasm, therebyproducing an enriched population of human RPE cells.
 2. The method ofclaim 1, wherein said culturing in step (b) comprises culturing themultilayer population of hES cells in media lacking exogenously addedFGF.
 3. The method of claim 1, wherein said culturing in step (b)comprises culturing the multilayer population of hES cells in medialacking exogenously added FGF and lacking exogenously added LIF.
 4. Themethod of claim 1, wherein said culturing in step (b) comprisesculturing the hES cells in media lacking exogenously added FGF andlacking exogenously added LIF and lacking exogenously added PLASMANATE®.5. The method of claim 1, wherein the duration of culturing in step (b)is about 6 weeks.
 6. The method of claim 1, wherein the duration ofculturing in step (b) is between about 6 weeks and about 8 weeks.
 7. Themethod of claim 1, wherein the duration of culturing in step (b) isbetween about 3 months and about 5 months.
 8. The method of claim 1,wherein the cell monolayer of step (d) contains cells that are Pax6−,bestrophin+, CRALBP+, PEDF+, and express RPE65.
 9. The method of claim1, wherein the cell monolayer of step (d) contains cells that are Pax6−,bestrophin+, CRALBP+, PEDF+, and express RPE65, and have the absence ofat least one ES cell marker selected from the group consisting of Oct4and Sox2.
 10. The method of claim 1, wherein prior to step (b) saidmultilayer population of hES cells are cultured in the presence ofexogenously added FGF and a fibroblast feeder layer.
 11. The method ofclaim 1, wherein prior to step (b) said multilayer population of hEScells are cultured the presence of exogenously added FGF and LIF and afibroblast feeder layer.
 12. The method of claim 1, wherein prior tostep (b) said multilayer population of hES cells are cultured thepresence of exogenously added FGF, PLASMANATE® and a fibroblast feederlayer.
 13. A method for producing an enriched population of humanretinal pigment epithelium (RPE) cells, the method comprising: (a)providing a culture of human ES (hES) cells; (b) culturing the hES cellsto produce one or more embryoid bodies; (c) culturing said one or moreembryoid bodies for a sufficient time for the appearance of putativehuman RPE cells within at least one of said one or more embryoid bodies,wherein said putative human RPE cells comprise brown pigment dispersedwithin their cytoplasm, whereby one or more embryoid bodies containingputative human RPE cells are formed; (d) selecting and dissociating oneor more of said embryoid bodies containing putative human RPE cells fromthe culture of step (c) to obtain human RPE cells; and (e) culturingsaid human RPE cells obtained in step (d) to form a cell monolayercontaining cells that are Pax6− and bestrophin+ and exhibit acharacteristic cobblestone, polygonal, epithelial-like appearance andcomprise brown pigment dispersed within their cytoplasm, therebyproducing an enriched population of human RPE cells.
 14. The method ofclaim 13, wherein the culturing of one of more embryoid bodies in step(c) comprises culturing the embryoid bodies in media lacking exogenouslyadded FGF.
 15. The method of claim 13, wherein the culturing of one ofmore embryoid bodies in step (c) comprises culturing the embryoid bodiesin media lacking exogenously added FGF and lacking exogenously addedLIF.
 16. The method of claim 13, wherein the culturing of one or moreembryoid bodies in step (c) comprises culturing the embryoid bodies inmedia lacking exogenously added FGF and lacking exogenously added LIFand lacking exogenously added PLASMANATE®.
 17. The method of claim 13,wherein the duration of culturing in step (c) is about 6 weeks.
 18. Themethod of claim 13, wherein the duration of culturing in step (c) isbetween about 6 weeks and about 8 weeks.
 19. The method of claim 13,wherein the duration of culturing in step (c) is between about 3 monthsand about 5 months.
 20. The method of claim 13, wherein the cellmonolayer of step (e) contains cells that are Pax6−, bestrophin+,CRALBP+, PEDF+, and express RPE65.
 21. The method of claim 13, whereinthe cell monolayer of step (e) contains cells that are Pax6−,bestrophin+, CRALBP+, PEDF+, and express RPE65, and have the absence ofat least one ES cell marker selected from the group consisting of Oct4and Sox2.
 22. A method for producing an enriched population of humanretinal pigment epithelium (RPE) cells, the method comprising: (a)providing a multilayer population of human embryonic stem (hES) cells,wherein said multilayer population of hES cells have been cultured inmedia containing exogenously added FGF and a fibroblast feeder layer;(b) culturing said multilayer population of hES cells under conditionsthat do not maintain the undifferentiated state of said hES cells for asufficient time for the appearance of putative human RPE cells, whereinsaid putative human RPE cells comprise brown pigment dispersed withintheir cytoplasm, wherein said conditions that do not maintain theundifferentiated state of said hES cells comprise media lackingexogenously added FGF and the duration of culturing is at least 6 weeks;(c) selecting one or more of said putative human RPE cells from theculture of step (b) to obtain human RPE cells; and (d) culturing saidhuman RPE cells selected in step (c) to form a cell monolayer containingcells that are Pax6−, bestrophin+, CRALBP+, PEDF+, and express RPE65,have the absence of at least one ES cell marker selected from the groupconsisting of Oct4 and Sox2, and exhibit a characteristic cobblestone,polygonal, epithelial-like appearance and comprise brown pigmentdispersed within their cytoplasm, wherein during said culturing thecultured cells temporarily lose their epithelial appearance andpigmentation after plating, and then regain their epithelial appearanceand pigmentation upon further culturing, thereby producing an enrichedpopulation of human RPE cells.
 23. The method of claim 22, wherein saidculturing in step (b) comprises culturing the multilayer population ofhES cells in media lacking exogenously added FGF and lacking exogenouslyadded LIF.
 24. The method of claim 22, wherein said culturing in step(b) comprises culturing the multilayer population of hES cells in medialacking exogenously added FGF and lacking exogenously added LIF andlacking exogenously added PLASMANATE®.
 25. The method of claim 22,wherein the duration of culturing in step (b) is between about 6 weeksand about 8 weeks.
 26. The method of claim 22, wherein the duration ofculturing in step (b) is between about 3 months and about 5 months. 27.The method of claim 22, wherein said media containing exogenously addedFGF in step (a) further comprises exogenously added LIF.
 28. The methodof claim 22, wherein said media containing exogenously added FGF in step(a) further comprises exogenously added PLASMANATE®.
 29. A method forproducing an enriched population of human retinal pigment epithelium(RPE) cells, the method comprising: (a) providing a culture of humanembryonic stem (hES) cells; (b) culturing the hES cells to produce oneor more embryoid bodies, wherein said one or more embryoid bodies or thecells from which said one or more embryoid bodies are formed have beencultured in media containing exogenously added FGF; (c) culturing saidone or more embryoid bodies for a sufficient time for the appearance ofputative human RPE cells within at least one of said one or moreembryoid bodies, wherein said putative human RPE cells comprise brownpigment dispersed within their cytoplasm, wherein the one or moreembryoid bodies are cultured in a media lacking exogenously added FGF,and the duration of culturing is at least 6 weeks, whereby one or moreembryoid bodies containing putative human RPE cells are formed; (d)selecting and dissociating one or more of said embryoid bodiescontaining putative human RPE cells from the culture of step (c) toobtain human RPE cells; and (e) culturing said human RPE cells obtainedin step (d) to form a cell monolayer containing cells that are Pax6−,bestrophin+, CRALBP+, PEDF+, and express RPE65, have the absence of atleast one ES cell marker selected from the group consisting of Oct4 andSox2, and exhibit a characteristic cobblestone, polygonal,epithelial-like appearance and comprise brown pigment dispersed withintheir cytoplasm, wherein during said culturing the cultured cellstemporarily lose their epithelial appearance and pigmentation afterplating, and then regain their epithelial appearance and pigmentationupon further culturing, thereby producing an enriched population ofhuman RPE cells.
 30. The method of claim 29, wherein said culturing instep (c) comprises culturing the embryoid bodies in media lackingexogenously added FGF and lacking exogenously added LIF.
 31. The methodof claim 29, wherein said culturing in step (c) comprises culturing theembryoid bodies in media lacking exogenously added FGF and lackingexogenously added LIF and lacking exogenously added PLASMANATE®.
 32. Themethod of claim 29, wherein the duration of culturing in step (c) isbetween about 6 weeks and about 8 weeks.
 33. The method of claim 29,wherein the duration of culturing in step (c) is between about 3 monthsand about 5 months.
 34. The method of claim 29, wherein said mediacontaining exogenously added FGF in step (b) further comprisesexogenously added LIF.
 35. The method of claim 29, wherein said mediacontaining exogenously added FGF in step (b) further comprisesexogenously added PLASMANATE®.